Since 1967, my research goal is focused on the issue of mechanism of transmitter released from sympathetic neuroeffector organs. In 1980, tissue culture technique was adopted with a hope that a pure population of sympathetic neurons (SN) would be available to study the molecular mechanism of transmitter release by determining the role of calcium ions (Ca) and other second messengers. Toward such a goal, several noradrenergic properties of cultured SN were established over the past 11 funding years. During the next support period we intend to probe further into the mechanism of release of norepinephrine (NE) by applying techniques not available in the past. Four major aspects of neurosecretion will be covered in the proposed research plan. 1. Physiological and pharmacological characteristics of 3H-NE release from two specialized regions of SN (I.E. cell body and nerve terminal or growth cone) will be evaluated. 2. Movements of Ca will be monitored in the cell body and the growth cones to know how these regions handle Ca and respond to agents that modulate 3H-NE release by affecting Ca. 3. Involvement of second messengers will be investigated on 3H-NE release and Ca movements to know how protein kinase C, inositol 1,4,5-triphosphate and cAMP cross-communicate to modulate release and neuronal Ca. 4. Trophic influence of heart cells on 3H_NE release and Ga-movements will be determined to know if heart cells modulate the release by affecting Ga movements in SN. Since the current proposal includes methods that are capable of giving more direct information, it is anticipated that upon completion of the present study a comprehensive picture will emerge concerning the role of Ca and other second messengers in the transmitter release process. Most important, these studies will provide new insights regarding the trophic influence of heart cells on their fields of innervation during growth and development. A number of preliminary results support the feasibility of the proposed research plan.